Microbial exopolysaccharide and uses thereof

ABSTRACT

A novel microorganism producing a nontoxic, non-antigenic exopolysaccharide is taught. The exopolysaccharide has neutral sugars migrating at the same rate as mannose, fucose, fructose and galactose, acidic sugars migrating at the same rate as fucose and amine sugars migrating at the same rate as glucose and fucose, and wherein the ratio of galactose:fucose:glucose:mannose is about 1:2:3:6. The microbe and the exopolysaccharide have uses as a biofilm in geologic applications and have several consumer uses as food and drug polymers and use as a plasma extender.

CROSS-REFERENCES TO RELATED APPLICATIONS

This Application for Patent claims the benefit of priority from, andhereby incorporates by reference the entire disclosure of, co-pendingU.S. Provisional Application for Patent Ser. No. 60/161,588 filed Oct.26, 1999 and Ser. No. 60/161,391, filed Oct. 26, 1999.

TECHNICAL FIELD OF THE INVENTION

The present invention relates in general to a novel non-pathogenicmicrobe that produces a nontoxic, non-antigenic exopolysaccharide. Theuse of the microbe and exopolysaccharide in environmental engineering,agricultural, geologic, consumer and medical applications is described.

BACKGROUND OF THE INVENTION

The invention pertains to a novel non-pathogenic microbe that produces anon-toxic, non-antigenic exopolysaccharide. The exopolysaccharide can beused as a biofilm in environmental engineering and agriculturalapplications and as a filler or polymer in consumer and medicalapplications. Biofilm applications are described first, then particularmedical applications are described.

The term “biofilm” is used to describe an organic material that includesmicroorganisms embedded in a polymer matrix of their own making. Thematrix consists largely of exopolysaccharides and is a tough, elastic,mucoidal material that adheres strongly to soil particles. Growth of abiofilm in a sandy soil is achieved by injecting a bacterial andnutrient solution into soil specimens. The resulting biofilm treatmentis used to clog soil pores, thereby reducing the ability of the soil totransmit fluids.

Examples of biofilms are produced by certain strains of Klebsiellapneumoniae and Pseudomonas species. A problem with the use of K.pneumoniae is that Klebsiella is a genus that includes a number of humanpathogens. Furthermore, the pathogenicity of K. pneumoniae itself isassociated with its ability to create a mucoidal exopolysaccharide usedin attachment and colonization that helps the pathogen evade both thenon-specific and specific immune clearing defensive mechanisms.

Another example of a biofilm is described in U.S. Pat. No. 4,800,959, byCosterton, which discloses the use of a microbial process forselectively plugging a subterranean formation. In the process taught, ahighly permeable stratum or zone in a subterranean reservoir is pluggedusing Klebsiella or Pseudomonas bacteria that were starved to reducetheir size prior to being injected into the target zone. The bacteriaregain full cell size, proliferate and commence production ofbiofilm-forming exopolysaccharides upon exposure to minimal nutrientcontaining media. The biofilm produced by these bacteria selectivelyseal off the high permeability zones of a formation and reduce aqueousflow through the zone.

In addition to the above described biofilm uses, there has been a needfor perfusion solutions and blood substitutes. Currently available andapproved compounds, however, have so far failed to meet the increasingdemands on our blood provider system. A number of blood substitutes havebeen developed over the last few years to attempt to meet the increasingdemand for blood, blood substitutes and plasma expanders. Unfortunately,many of the plasma expanders that are currently in use fail as the smallmolecules on which they depend to provide osmotic pressure readilytraverse capillary beds as a consequence of the negative osmoticpressure found in post-arterial capillary beds. The loss of osmoticpotential, makes the long-term use of current plasma expanders formaintaining proper ionic or fluid balance or plasma volume in amammalian subject unsatisfactory.

Those blood substitutes that have an impermeable substance to maintainvolume use human serum albumin or a mixture of plasma proteins as theoncotic agent. These substitute plasma proteins depend on the same bloodand plasma supply as our current blood provider system, thereforefailing to meet the increased demand for these products.

A number of patents have issued to Segall that are directed to blood andplasma substitutes. U.S. Pat. No. 4,923,442, and the reissue thereof,discloses a number of solutions used in blood substitution of livingsubjects all of which include at least some concentration of acardioplegia agent, usually potassium ion. U.S. Pat. No. 4,923,442discloses surgical methods, particularly in respect to instrumentplacement and the control of pulmonary wedge pressure generallyapplicable to perfusion of subjects. U.S. Pat. No. 5,130,230 discloses ablood substitute that may be used as a system of solutions in which anumber of solutions, are used sequentially to completely replace theblood of living subjects. U.S. Pat. No. 5,130,230 discloses that theblood substitute comprises “an aqueous solution of electrolytes atphysiological concentration, a macromolecular oncotic agent, abiological buffer having a buffering capacity in the range ofphysiological pH, simple nutritive sugar or sugars, and magnesium ion ina concentration sufficient to substitute for the flux of calcium acrosscell membranes.”

In addition to the patented inventions described above, a number ofcommercially available products have been used for the treatment ofhypovolemic patients. These include: HESPAN™ (6% hetastarch in 0.9%sodium chloride injection, PENTASPAN™ (10% pentastarch in 0.9% sodiumchloride injection [both by DUPONT PHARMACEUTICALS™, Wilmington Del.]),MACRODEX™ (6% dextran 70 in 5% dextrose injection or 6% dextran 70 in0.9% sodium chloride injection [PHARMCIA, INC.™, Piscataway, N.J.]) andRHEOMACRODEX™ (10% dextran 40 in 5% dextrose injection or 10% dextran 40in 0.9% sodium chloride injection [PHARMACIA, INC.™, Piscataway, N.J.]).All of these products, however, depend on compounds that are polymericand that often dissociate or are broken down by natural physiologicenzymes with time. Alternatively, bacteria may take advantage of thesenewly supplied nutrient sources, causing severe septicemia in patientsthat are infected by pathogens at the time of injury. Thus, a needremains for a better oncotic agent.

SUMMARY OF THE INVENTION

The newly discovered bacterium LAB-1, deposited at ATCC No. PTA-2500,possesses a number of potential commercial biofilm applications. Theseinclude, but are not limited to: (1) subsurface biofilm cutoff wallformation; (2) subsurface liners that include compacted, biofilm treatedsoil; (3) in-situ biofilm liners; (4) barriers made by treatinggeotextiles with biofilm materials; (5) improved ability of sand toretain moisture; (6) reclamation of poor soils and conversion intoagriculture land; (7) significantly increased soil biomass in the formof polymers that function as a nutrient supply for plant growth and/orhelp retain nutrients and water; and (8) providing cohesion to otherwisecohesionless soils (such as sand dunes), thus making the soil moreresistant to erosion by wind and/or water.

It has been found that the prior art methods and biofilms fail toprovide biologically and environmentally safe and efficacious water,soil and waste retention characteristics. A significant problem withexisting technology is the pathogenicity of the bacteria used to producethe biofilms. The present invention, therefore, is directed to anon-pathogenic bacterium that produces a biofilm made ofexopolysaccharide that is essentially made of neutral sugars thatmigrate at the same rate as: mannose, fucose, fructose and galactose,acidic sugars that migrate at the same rate as fucose and amine sugarsthat migrate at the same rate as glucose and fucose.

More particularly, the bacterium is a LAB-1 strain. The biofilmproducing bacterium may be further defined as being capable of growthbetween about pH 4 and 11 and between about 15° and 45° C. The LAB-1strain is capable of growth in minimal growth media, or may be grown inan aqueous nutrient medium that includes yeast, peptone and mineral saltingredients. LAB-1 is a gram-negative, rod-shaped bacterium of about0.2×0.8 μm that secretes the exopolysaccharide described herein.

In one embodiment of the present invention, the LAB-1 strain is used inplugging a permeable subterranean stratum by providing LAB-1 bacteria ina nutrient-containing solution into the target stratum. Thenutrient-containing solution is generally adapted to provide substantialand uniform growth conditions for the LAB-1. Sufficient biofilm isproduced under these conditions to effectively plug the stratum. Forexample, the bacterium in situ can yield a saturated hydraulicconductivity equal to or less than 1.5×10⁻⁵ cm/sec, equal to or lessthan 1.0×10⁻⁷ cm/sec or even equal to or less than 1.5×10⁻⁸ cm/sec.

Alternatively, the bacteria may be preincubated in culture in an aqueoussuspension medium with agitation for an incubation period sufficient toinitiate bacterial exopolysaccharide production before injection intothe stratum. The method of plugging the subterranean stratum may alsoinclude draining nutrient deficient suspension medium from thereservoir, and recharging the reservoir with aqueous nutrient medium tomaintain bacterial growth for an elapsed time period sufficient toestablish a biofilm of prescribed saturated hydraulic conductivity. Thedraining and recharging steps with aqueous nutrient medium may beconducted at least once every 48 hours of elapsed time period. The stepof pre-incubating the bacteria may be, e.g., for at least about 72hours. These growth conditions permit for the establishment of a biofilmhaving a population between about 10⁵-10¹⁵ bacterial Colony FormingUnits per square centimeter on a slide surface.

The biofilm may be used to plug open conduits, deposited in a subsurfacebiofilm cutoff wall, used to enhance the water retaining ability ofsubsurface liners or even for improving the water retention capabilitiesof compacted, semi-compacted or loosened biofilm treated soil. When usedin a liner, the biofilm may be deposited in-situ. The biofilm may alsobe used along with and/or to enhance environmental barriers by treatinggeotextiles with the biofilm.

Another important aspect of this polymer is its lack of antigenicity andtoxicity in an animal system. This suggests several consumer/medicalapplications, including: (1) use a food additive or food thickening orfiller agent; (2) use as plasma expander; (3) use in polymer industry;(4) use as chromatography matrix support for purification of chemicals;(5) use in scientific research as suspension solution instead of ficolland the like; (6) use in determining the gene content of the organism,especially those coding for the biosynthesis of the exopolysaccharidepolymer; (7) use of the polymer materials in the cosmetic field; (8) useto augment insect or animal diets; (9) use as an additive in tissueculture media; (10) for use as a semi-solid to solid matrix, e.g, gelelectrophoresis; (11) for use as an additive in toothpaste, ointments,creams and lotions; (12) for mixing with dyes, stains, paints andvarnishes; (13) for inclusion in dialysis; (14) for use in compositematerials, e.g., bricks, tile, mortars; (15) for use as part of asealant; (16) viscosity modifier for oils, waxes & greases; (17) use asa filler, thickener or extender in pharmaceutical preparations; (18) useof the polymer in bioscaffolding applications, including wound-healingapplications; and (19) use as a bacteriostatic (biostat) agent toinhibit or at least fail to support bacterial growth, and even possiblyas a biocide.

In particular, a compound is needed for use as a plasma extender thatserves to increase blood volume and that is impermeable at bloodcapillaries. The compound must not readily dissociate or be rapidlybroken down by natural physiologic enzymes with time. Furthermore, thecompound and its use as a plasma expander must not provide bacteria withan exogenous nutrient source, which may lead to accentuating alreadysevere septicemia in patients that are infected by pathogens at the timeof the injury that is causing hypovolemia.

More particularly, the present invention is an exopolysaccharideproduced by the LAB-1 bacterial stain. The exopolysaccharaide does notappear to easily support bacterial growth. This was determined bytesting the ability of E. coli or B. indica to grow on theexopolysaccharide and no growth was observed. Further, theexopolysaccharide is not antigenic as tested by injection into mice.Thus, the product appears to satisfy some of the basic parametersrequired for a plasma expander.

The exopolysaccharide is secreted into the cell culture medium andcollected for use in, e.g., a plasma expander. When used as a plasmaexpander alone, or in combination with other elements, theexopolysaccharide will be provided in an isotonic solution. In oneembodiment, a blood-free plasma expander and blood substitute for use ina subject in need thereof includes a single solution with at least twowater soluble oncotic agents, one of which is a water solublepolysaccharide oncotic agent and one of which is serum albumin, whereinthe exopolysaccharide consisting essentially of mannose, fucose,fructose and galactose, acidic fucose and amine containing glucose andfucose.

The plasma expander and blood substitute may have a ratio of watersoluble exopolysaccharide oncotic agent to serum albumin between 1:1 and1:2, weight to weight. The combined percentage of water-solubleexopolysaccharide oncotic agent and serum albumin in a solution of theplasma expander and blood substitute may be in the range of betweenabout 4%-6% weight to volume.

The plasma expander and blood substitute may also include a number ofcations, alone or in combination. For example, the cations may beprovided in the following concentrations: Na⁺ at 110 to 120 mEq/1, Ca⁺⁺at about 5 mEq/1, K⁺ at 0 to 3 mEq/1, and Mg⁺⁺ at 0 to 0.9 mEq/1. Thesecations may be supplied as dissolved chloride salts. The plasma expanderand blood substitute may also include at least one buffer, for example,a lactate and/or bicarbonate buffer. When buffered, the plasma expanderwill generally be a biological buffer having a buffering capacity in thepH range of about 6.8 to 7.8.

When used in hypovolemic patients, e.g., those that have lost a largevolume of blood due to trauma, additional agents may be included in theplasma expander to aid in recovery. Such agents may include, Vitamin Kin a concentration of about 1-4 mg/1, amylase, clotting factors, t-PA oreven erythropoietin.

In non-medical uses, the exopolysaccharide of the present invention maybe used as a chromatography matrix support for purification ofchemicals. One such use will be as a suspension solution for use incentrifugation. The exopolysaccharide may even be used in solution as asuspension solution for use in size separation.

The present invention may also be used as a biologically stable,non-toxic material for use in coated plates for a number of biologicaland analytical uses. Examples of such uses include the coating of tissueculture plates for maintaining the growth, in vitro, of cells. Cellsthat may be grown on the surface of the exopolysaccharide includeprokaryotic and eukaryotic cells. In an analytical setting, theexopolysaccharide disclosed herein may be used as a coating forinstrumentation, such as biosensors, that require the maintenance of abiologically compatible environment.

A more complete appreciation of the present invention and the scopethereof can be obtained from the accompanying drawings which are brieflysummarized below, the following detailed description of thepresently-preferred embodiments of the invention, and the appendedclaims.

BRIEF DESCRIPTION OF THE DRAWINGS

A more complete understanding of the method and apparatus of the presentinvention may be obtained by reference to the following DetailedDescription when taken in conjunction with the accompanying Drawingswherein:

FIG. 1 is a photograph of Gram stained LAB-1 at 100×magnification.

FIG. 2 is a Coomassie stained SDS-PAGE gel of the total protein contentof LAB-1 and B. indicia grown on solid culture.

FIG. 3 is a Coomassie stained SDS-PAGE gel of the total protein contentof LAB-1 and B. indicia grown in liquid culture.

FIG. 4 is the data from gas chromatography of fatty acids in LAB-1.

FIG. 5 is a FACE gel showing the sugars identified in theexopolysaccharide produced by LAB-1. Lanes: 1—MONO Ladder Standard 2(100 pmol ea. monosaccharide); 2—Amine hydrolysis reaction products; 3[S]—MONO Ladder Standard 2 (100 pmol ea. monosaccharide; scanned fortrace shown in [S] Scan); 4—Neutral hydrolysis reaction products;5—Sialic acid hydrolysis reaction products; 6—NANA labeling control 1(100 pmol); 7—MONO composition control; 8—MONO Ladder Standard 2 (100pmol ea. monosaccharide).

FIG. 6 is a MALDI trace of the exopolysaccharide produced by LAB-1.

FIG. 7 is a drawing of a cross sectional view of a barrier created withthe biofilm of the present invention.

FIG. 8 is a graph of the hydraulic conductivity versus time of a LAB-1containing biofilm.

DETAILED DESCRIPTION OF THE PRESENTLY PREFERRED EXEMPLARY EMBODIMENTS

The present invention will now be described more fully hereinafter withreference to the accompanying drawings,.in which preferred embodimentsof the invention are shown. This invention may, however, be embodied inmany different forms and should not be construed as limited to theembodiments set forth herein; rather, these embodiments are provided sothat this disclosure will be thorough and complete, and will fullyconvey the scope of the invention to those skilled in the art.

EXAMPLE 1 Characterization of LAB-1 Organism and Exopolysaccharide

Samples of LAB-1 have been deposited under ATCC No. PTA-2500. LAB-1 is aGram-negative, rod-shaped bacterium isolated by the present inventorfrom constructed soil samples in the state of Wyoming. The soil samplewas constructed by J. Turner at the University Wyoming and contained acontaminant in a background of a Beijerinckia indica. The contaminant,LAB-1, was isolated and studied because of its excessive slimeproduction.

A variety of biochemical tests were performed in order to identify thespecies and the genus of organism. The tests included: assessment ofgrowth conditions, culture appearance, cell appearance and stainingcharacteristics, optimal temperature and pH growth range, oxygenrequirements, antibiotic sensitivity testing, tests for catalase andoxidase (using TAXOS N™ disks), amino acid utilization as carbon source,nitrogen fixation, motility test, and a variety of commerciallyavailable tests from MICRO-ID™, OXYFERM™, ENTEROTUBE™ and BIOLOG GNMICROPLATE™.

Because the organism was a contaminant in soil amended with a B. indicaculture, B. indica and the LAB-1 organism were grown on plates and totalprotein profiles were compared by SDS-PAGE. Fatty acid analysis wasperformed by Microbial ID (MIDI) of Newark, Del. who used the highresolution MIDI SHERLOCK SYSTEM™ to identify fatty acids with highresolution gas chromatography. The same laboratory also sequenced the16S gene and compared it against the proprietary MICROSEQ™: database (PEAPPLIED BIOSYSTEM™)

The exopolysaccharide was further analyzed by GLYKO™ (NOVATO™,California) fluorophore-assisted carbohydrate electrophoresis (FACE),and matrix-assisted laser desorption/ionization mass spectrometry(MALDI). Immunogenicity of the exopolysaccharide was also tested inmice.

Colonies of LAB-1 on solid medium were irregular with an undulate edge.They exhibited convex elevation, a smooth glistening surface, were whitein color and translucent to opaque. The consistency of the colonies wasthat of a very tenacious and elastic slime. The LAB-1 exopolysaccharideexhibits tremendous tenacity, extending without breakage when pulledwith a glass rod over a foot. Due to the production of thisexopolysaccharide, it was found difficult to lift the colonies from agarplates.

AB13 culture medium may be used to grow the LAB-1 strain and is made asfollows: Per liter of water add 20 g glucose, 1 g NaCl, 1 g yeastextract or 2 g NaNO₃, 8 g K₂HPO₄, 0.2 g KH₂PO₄, 0.5 g MgSO₄, and 150 μl5% FeCl₂. To make solid medium, 15 g of agar are added to the medium.This simplified end medium makes large-scale production of thepolysaccharide polymer affordable.

Growth in liquid AB13 medium was perfuse and the turbidity was dense anduniform. When the LAB-1 bacterium is grown in liquid medium, the entiresolution becomes very viscous with a consistency ranging from that ofheavy corn syrup to that of egg whites, depending on the stage ofgrowth. Cultures grown without shaking showed a flocculent deposit alongwith smooth surface growth. The cultures had a very distinct odor,somewhat sweet smelling, but not pleasant.

The bacteria were bacilliary with rounded ends and parallel sides andwere determined to be Gram negative. There were some irregularitiesobserved in the cell population in cell size, due mainly to lengthdifferences, but the average size was about 0.2×0.8 μm. The arrangementof cell packets seemed to be irregular, although a large percentage ofcells were aligned side-to-side. The clumping of cells was believed tobe due to the tenacious slime layer. The exopolysaccharide could be seenin Gram staining as a light cloud surrounding the cells. FIG. 1 showsthe Gram stained cells at 100×. The spore stain showed only redvegetative cells, no spores were observed. The capsule stain showed nocapsules, but rather an indefinite exopolysaccharide surrounding thecells. Observation of the stab culture showed that the organism wasmotile.

Colony morphology on plates grown at 26°, 30°, 37° C. and 45° C. was thesame, although growth was optimal at 37° C. Elasticity of the slimelayer was also unchanged. Growth was unchanged in liquid AB13 culturesranging from pH 4, 9 and 11. Growth was not observed at pH 2. Colonymorphology on plates was also unchanged over this pH range. The LAB-1organism was determined to be a facultative anaerobe.

Various biochemical test results are summarized in the following tables:

TABLE 1 Morphology of LAB-1 Morphological Characteristics Results Gramreaction negative Cell Shape bacilli Spores none Growth temp. 26-37° C.pH range 4-11 Growth in peptone + Motility +

TABLE 2 Antibiotic Profile of LAB-1 Antibiotic Susceptibility ResultsAmikacin sensitive Ampicillin resistant Erythromycin resistant Neomycinsensitive Novobiocin sensitive Penicillin resistant Polymyxin Bsensitive Streptomycin resistant Taxos A resistant Tetracyclinesensitive

TABLE 3 Biochemistry of LAB-1 Biochemical Properties Results Adonitolfermentation + Ammonification + Arabinose fermentation + Argininedihydrolase + Beta-galactosidase − Catalase + Citrate utilization +Cytochrome oxidase − Esculin hydrolysis − Glucose fermentation + Aerobicglucose + Anaerobic glucose + Hydrogen sulfide − Indole (tryptophanase)− Inositol fermentation + Lactose fermentation + Lysine decarboxylase −Malonate utilization − Maltose oxidation + Mannitol oxidation + N2 gasproduction − Nitrate reductase − Nitrogen fixation − Ornithinedecarboxylase − Phenylalanine deaminase − Sorbitol fermentation +Sucrose oxidation − Urease + VP + Xylose oxidation +

TABLE 4 Carbon Utilization of LAB-1 Carbon Utilization ResultA-cyclodextrin + Dextrin + Glycogen + Tween 40 + Tween 80 + N-acetylgalactosamine + N-acetyl-D-glucosamine + Adonitol + L-arabinose +D-arabitol + D-cellobiose + I-erythritol − D-fructose + L-fucose +D-galactose + Gentiobiose +/− A-D-glucose +/− M-inositol + A-D-lactose +Lactulose + Maltose + D-mannitol +/− D-mannose + D-melibiose +B-methyl-d-glucoside + D-psicose + D-raffinose + L-rhamnose +D-sorbitol + Sucrose + D-trehalose + Turanose + Xylitol + Methylpyruvate + Methyl succinate + Acetic acid + Cis-aconitic acid + Citricacid + Formic acid + D-galactonic acid + lactone D-galacturonic acid +D-gluconic acid + D-glucosaminic acid + D-glucuronic acid +A-hydroxybutyric acid − B-hydroxybutyric acid + G-hydroxybutyric acid −P-hydroxphenyl acetic acid + Itaconic acid − A-keto-butyric acid −A-keto-glutaric acid − A-keto-valeric acid − D, L-lactic acid + Malonicacid + Propionic acid − Quinic acid + D-saccharic acid + Sebacic acid −Succinic acid + Bromo Succinic acid + Succinamic acid +/−Glucuronamide + Alaninamide +/− D-alanine + L-alanine +L-alanyl-glycine + L-asparagine + L-aspartic acid + L-glutamic acid +Glycyl-L-aspartic acid + Glycyl-L-glutamic acid + L-histidine +Hydroxy-L-proline + L-leucine − L-ornithine − L-phenylalanine −L-proline + L-pyroglutamic acid − D-serine + L-serine + L-threonine −Carnitine − G-amino Butyric acid + Urocanic acid − Inosine + Uridine −Thymidine + Phenylethylamine − Putrescine − 2-amino ethanol −2,3-butanediol − Glycerol + D,L-A- glycerol phosphate +Glucose-1-phosphate + Glucose-6-phosphate +

Bacteriological information was compiled into the chart shown in Tables1 through 4. This information was used to search the 9^(th) edition(1994) of Bergey's Manual of Determinative Bacteriology. The Bergey'sprofiles of all Gram negative facultative anaerobic bacilli wereresearched in an attempt to find potential matches. Special attentionwas paid to members of the genus Klebsiella because this was the closestmatch provided by BIOLOG™, and, to the genus Beijerinckia because LAB-1was isolated as a contaminant amongst a lawn of Beijerinckia colonies.

Although the information contained in Tables 1-4 was consideredvaluable, this particular method of exploration was quickly proven to beinconclusive. No exact matches were identified, and, the partial matchesfound were too numerous to consider them all. Therefore, other moreconcrete methods of identification were utilized.

Because it was entirely possible that LAB-1 could have been aBeijerinckia species, protein profiles of each organism were compared.For the gel in FIG. 2, the colonies of each organism were harvested fromAB13 plates and sheared by vortexing with glass beads for five minutes.One volume of running buffer and one volume of bromophenol blue trackingdye was added to the lysate, and the result boiled for five minutes.After a brief spin, the samples were overlaid twice with powderedsucrose to remove cell debris and 100 μl were loaded onto a 4.5%stacking, 10% resolving polyacrylamide gel and run at 30-35 mAmp.

Samples used for FIG. 3 were grown in liquid AB13 culture. One ml washarvested by pelleting for five minutes, washed with 1% NaCl and againwith dH₂O. Pellets were resuspended in 180 μl of 50 mM Tris, and 10 mMEDTA, pH 8.0. Sixty μl of PSS (4×PSS is 700 μl of 1 M Tris-HCl, pH 6.8,4.3 g sucrose, 4 mg bromophenol blue, 1.45 μl of 20% SDS, 44 μl of 0.5 MEDTA, 10 mg DTT and dH₂O to 10 ml) were added to the cells and themixture was boiled for five minutes. Samples were overlaid with powderedsucrose to remove cell debris and run as above.

The Coomassie blue stained SDS-PAGE gels shown in FIGS. 2 and 3 clearlydemonstrate that there are dissimilarities between the two organisms. Itwas concluded that the unknown organism LAB-1 was not a Beijerinckiaspecies.

Fatty acid analysis by gas chromatography by MIDI LABS™, Newark, Del.,was employed to identify the organism. The growth conditions werestandardized, the MIDI SHERLOCK SYSTEM™ was fully automated, and thedata compared against proprietary databases containing the fatty acidprofiles of more than 1,900 bacteria. The results are shown in FIG. 4.The best match, Kluyvera cryocrescens, had a similarity of 0.835. Thismatch would have been considered very good if there had been aseparation of at least 0.100 between this first choice and the secondchoice, Enterobacter taylorae, with a similarity of 0.753.

Using these two species, reference was made to the 9^(th) edition (1994)of Bergey's Manual of Determinative Bacteriology to compare theircharacteristics with those listed in Tables 1-4.. Also compared was thethird choice, Kluyvera ascorbata. There were many key differences in thebiochemical profiles of LAB-1 and each of the three matches.

The properties found to be different amongst these four organisms areshown in Table 5. This information, along with the fatty acid analysisresults, led to the conclusion that the organism was not a member of thegenus Kluyvera.

TABLE 5 Differences between LAB-1 and K. cryocrescens, K. ascorbata, E.Taylorae LAB-1 K. cryo. K. ascorb. E. Taylorae Adonitol + − − −fermentation Arginine + − − + dihydrolase Esculin hydrolysis − + + +Indole − + + − (tryptophanase) Inositol + − − − fermentationLactose + + + − fermentation Lysine − − + − decarboxylaseMelibiose + + + − fermentation MR − + + − VP + − − + Nitrate reductase− + + + Ornithine − + + + decarboxylase Raffinose + + + − fermentationSorbitol + + + − fermentation Urease + − − −

The next logical step was to determine the 16S rRNA gene sequence. Thistechnique currently is the method of choice for identification purposes.The identification based on the 16S rRNA gene sequence was determined tobe Leclercia adecarboxylata. The difference in sequence homology betweenLAB-1 and L. adecarboxylata was only 0.59%. Stackebrandt & Goebel, INT'LJ. SYSTEM, BACTERIOL. 44: 846 (1994) would consider this a species levelmatch, however the confidence limits of the data obtained by MIDI LABS™allowed identification only at the genus level. When the biochemicalcharacteristics of LAB-1 and of L. adecarboxylata (9^(th) editionBergey's Manual of Determinative Bacteriology 1994) are compared, thereare yet again, numerous differences. The differences led to thequestions regarding this method.

TABLE 6 Differences between LAB-1 and L. adecarboxylata LAB-1 L.adecarb. Arginine dihydrolase + − Citrate utilization + − Esculinhydrolysis − + Glycerol utilization + − Indole (tryptophanase) − +Inositol fermentation + − Malonate utilization − + MR − + VP + − Nitratereductase − + Sorbitol fermentation + − Urease + −

Because the same laboratory (MIDI LABS™) produced two differentidentifications, the validity of both identities was suspect. However,16S rRNA gene sequencing is considered to be the most reliable methodcurrently available, and, therefore, the identification of Leclerciaadecarboxylata must be further investigated. With all of thediscrepancies that have been encountered, it is reasonable to concludethat this unknown organism (LAB-1) has not been previously identified.

Analysis of the exopolysaccharide produced by LAB-1 was by FACE (sugarcontent) and MALDI (size). Judging from the intensity of the bands shownon the FACE gel in FIG. 5, the amount of fucose present is approximatelytwice that of galactose; the amount of glucose is approximately 2.5times that of galactose; and, the amount of mannose is approximately 3times that of fucose. Therefore, the ratio ofgalactose:fucose:glucose:mannose is approximately 1:2:3:6. Furtherinvestigations regarding the nature and type of linkages and molecularweight determination of the polymer may be undertaken, as will be knownto those of skill in the art.

Results of the MALDI study of the exopolysaccharide are shown in FIG. 6.The data indicates that polymerization and depolymerization of thepolysaccharide occurred readily as evidenced by the large range ofmolecular weights found. All monosaccharides identified were neutralsugars that migrate at the same rate as: mannose, fucose, fructose andgalactose, acidic sugars that migrate at the same rate as fucose andamine sugars that migrate at the same rate as glucose and fucose. Allare six-carbon sugars. This fact makes it impossible to determine thecomposition of the polymer when only the molecular weight is known.Those substances with molecular weights below 180 are likely breakdownproducts of the polymer. The largest polymer, molecular weight of1066.38, was comprised of approximately six 6C sugars.

It was later discovered that the initial exopolysaccharide sampleanalyzed for immunogenicity in mice contained residual amino acids. Thesmall amount of protein present was from the yeast extract in the mediaand from dead cells. Even with these amino acids present, it wasdetermined that the exopolysaccharide was non-immunogenic, although avery small immunogenic reaction was observed. This reaction made itnecessary to further purify the exopolysaccharide so that it wasprotein-free.

It was determined that the best way to free the exopolysaccharide ofamino acids was to (i) modify the medium to contain no amino acids and(ii) limit growth of the organism to prevent cell death and breakdown.After implementing these two modifications, the exopolysaccharide wasfound to be free of protein (not shown). The protein-freeexopolysaccharide was provided to WASHINGTON BIOTECHNOLOGY™ [St Louis,Mo.] to perform more in-depth studies to determine immunogenicproperties. The results were gratifying and the exopolysaccharideimmunized rabbits maintained antibody titers of less that 1:100, ornon-detectable, for the entire 12 week experiment. In contrast, theinactivated cell-immunized rabbits reached antibody titers as high as1:1,638,400. These results are very encouraging because anon-immunogenic biopolymer with tremendous elasticity such as the onecharacterized in this study probably has numerous industrial,agricultural and biomedical applications.

Much information has been gathered about LAB-1. Unfortunately, itscomplete identity remains-indeterminate. Reliable identification methodshave been employed but the results do not agree with each other. It ishighly likely that this organism has not been previously identified.

More research is warranted before a definitive identification can bemade. Further studies should include direct comparison of LAB-1 withKlebsiella, Kluyvera cryocrescens and Leclercia adecarboxylata. Methodsimportant to compare these organisms include protein profiledeterminations and DNA analysis. Results obtained from these approacheswill provide good evidence of any phylogenetic relationships.

To further characterize the exopolysaccharide, the monosaccharidelinkages and branching of the polysaccharides should be determined.Also, it would be very useful to determine the nature of its overallpolymerization. The localization of the gene(s) coding for thepolysaccharides may be determined, as will be known to those of skill inthe art of molecular biology. Even further studies may be conducted toidentify LAB-1, and may include: chromosomal DNA fingerprinting, randomprimer PCR profiling, rRNA or other gene sequencing, determination ofthe G+C % content, lipid analysis and BIOLOG™ analysis (a morecomprehensive biochemical analysis). Also, detailed studies regardingthe chemistry of the polysaccharide will be completed.

EXAMPLE 2 Applications to Engineered Waste Containment and Treatment

The LAB-1 strain may be used to construct environmental biofilm barriersfor containment and treatment of contaminated soil and groundwater. Thepurpose of containment barriers is to control the transport of chemicalcontaminants from waste disposal facilities or from areas which havebecome contaminated by spills, industrial processes, illegal dumping orother sources. Several different types of barriers are possible,including the following: (1) subsurface biofilm cutoff wall; (2)subsurface liners consisting of compacted, biofilm treated soil; (3)in-situ biofilm liners; and (4) barriers made by treating geotextileswith biofilm.

The results disclosed herein demonstrate that soil hydraulicconductivity (k) may be reduced by several orders of magnitude by theaddition of the biofilm-producing bacterium disclosed herein. Thereductions of k obtained using the LAB-1 strain are sufficient to meetEnvironmental Protection Agency (EPA) criteria for barrier materials,defined as a k value of 10⁻⁷ cm/sec or less. The low hydraulicconductivity persists when the soil is permeated with a variety ofchemical solutions, suggesting that a biofilm barrier may be compatiblewith a wide range of contaminants. The biofilm disclosed herein may alsobe useful for controlling contaminant transport mechanisms, such asdiffusion, adsorption and biodegradation.

Solutions of biofilm and nutrient are pumped into the subsurface througha series of closely-spaced vertical wells. Formation of biofilm in thesoil around the wells causes a decrease in soil permeability anddecrease in contaminant transport sufficient to form an engineeredbarrier to contaminant migration. Specific design parameters such aswell depth and spacing, pumping pressures, composition of bacterial andnutrient solutions, and time of pumping, are site-specific and dependupon site geology, type and extent of subsurface contamination, groundwater conditions, and other variables which must be considered on acase-by-case basis.

FIG. 7 shows one use of the present invention for the formation ofsubsurface liners for the containment of wastes in engineered disposalfacilities, such as landfills. A landfill 10 is depicted incross-sectional view. Waste 12 is disposed within a subsurface liner 14.If the liner 14 is being placed during the creation of the landfill 10,a biofilm liner may be used prior to deposition of the liner 14. Inaddition, a containment wall may be erected that surrounds the wastesite, and additional layers of decontaminating biofilm barriers may beincluded.

In preexisting landfills, such as the one depicted in FIG. 7, waste mayleach in the form of a leachate 16 into 7 subsurface strata 18 and 20. Abiofilm barrier wall 28 is created that surrounds the waste 12 andcaptures the leachate 16. The biofilm barrier wall 28 is constructed soas to reach into strata 22, 24 into which the waste 12 does not leach.One advantage of the biofilm of the present invention is that it permitssuch remedial application to existing landfills that may be leaking andeven prevents leachate 16 from reaching a subterranean water layer 26.

Current technology for a biofilm barrier wall 28, for example, mayemploy fine-grained soils that are field compacted to achieve ahydraulic conductivity of less than 10⁻⁷ cm/sec (commonly referred to as“clay liners”). At many sites, such soil is not readily available andmust be transported from off-site, increasing substantially the cost ofcompacted soil liners. Using the LAB-1 strain of the present inventionsoil, containment conditions may be met by treating readily availablesoils with the biofilm in order to achieve the low hydraulicconductivity required for compacted soil liners.

One specific field of use for the LAB-1 biofilms is creating subsurfacebiofilm liners that include spreading untreated soil in loose(uncompacted) lifts using conventional soil spreading equipment (e.g.,bulldozers). Loose lifts will generally be 150 to 225 cm thick. Asolution that includes water, LAB-1, and nutrients are applied to thesoil, using, e.g., conventional equipment used to apply water to soil(e.g., a truck-mounted water tank with sprinkler hoses). The soil isthen compacted using conventional equipment (e.g., sheeps foot rollers)to achieve the specified density, typically resulting in a compactedlift thickness of 100 to 150 cm. The required number of lifts and linertotal thickness are site-specific design parameters which are determinedby analysis of contaminant transport and regulatory requirements forcontainment.

Compacted clay liners typically range from 0.6 to 1.3 meters thick. Theproposed procedure is similar to field construction of clay liners,except that the soil is treated with a biofilm-producing solution.Alternatively, solutions of strain LAB-1 and nutrients are injected intothe ground at a specified depth to create in situ biofilm liners. Thistype of liner is particularly useful at sites contaminated by accidentalspills. Alternatively, previously grown biofilm may be mixed directlyinto or onto the soil.

Subsurface liners may also be constructed by treating geotextiles withbiofilm. Geotextiles are generally made of synthetic fibers that areeither woven or matted together, yielding a porous fabric that is usedfor soil separation, reinforcement, filtration or drainage. Containmentbarriers can be created by spraying bitumen, rubber-bitumen or otherpolymeric mixtures into a properly deployed geotextile that contains theLAB-1 produced biofilm disclosed herein. One particular example for useof the LAB-1 biofilm is in the application of a liquid solutioncontaining strain LAB-1 and nutrients to geotextiles to clog the porespaces and reduce permeability, creating a barrier to flow.

EXAMPLE 3 Materials Soil and Bacteria

Soil used by the present inventors to analyze waste containmentcapability is a naturally occurring, easily attainable sand. Based onits grain size distribution and Atterberg limits, this soil isclassified as SM, or silty sand of low plasticity, in the Unified SoilClassification System. Permeability tests yield a saturated hydraulicconductivity (k) of approximately 1.5×10⁻⁵ cm/sec when compacted tomaximum dry density. This value of k would make the soil unsuitable foruse as a waste containment barrier. Initial studies indicated that kcould be reduced to values on the order of 10⁻⁸ to 10⁻⁷ cm/sec, which isin the range required for waste containment, by treating this soil withthe biofilm-producing bacterial strain LAB-1.

The operational procedure for use of the LAB-1 bacterium to form abiofilm that may be used to test water permeability may include thefollowing steps: (1) compacting soil into a cylindrical specimen whichis placed in a flexible wall permeameter, (.2) permeating the specimenwith a solution containing LAB-1, and (3) measuring the soil hydraulicconductivity while the specimen is permeated first with nutrientsolution, then by water, as is taught by Dennis M. L. and Turner, J. P.J. Geotechnical & Geoenvironmental Eng. 124: 120-127 (1988) (in which asimilar procedure was used with the bacterium B. indica).

Using the biofilm produced by the LAB-1 strain disclosed herein,hydraulic conductivity was reduced from k=1.5×10⁻⁵ to approximatelyk=5×10⁻⁸ cm/sec upon establishment of a plugging biofilm, which requiredpermeation with nutrient solution for approximately one week. Most ofthis decrease occurred within 1 to 2 days, during which the k wasreduced to less than 10⁻⁷ cm/sec.

FIG. 8 is a graph that shows hydraulic conductivity versus time for aspecimen treated with LAB-1. The low hydraulic conductivity persistedfor over 160 days, even though the nutrient-solution was discontinuedafter 6 days.

EXAMPLE 4 Applications to Atgriculture

Large areas of the earth include desert lands that are not arablewithout large-scale reclamation. Reclamation in the context of desertlands requires not only irrigation, but extensive soil modification. Theeconomical and social impact of successfully converting non-productivedesert land into productive agricultural land is enormous and providessignificant benefits to mankind.

The use of LAB-1 as a biologically and environmentally sound source ofsupport and nutrients for soil treatment improves the agriculturalproperties of sandy soils as described herein. Many naturally existingdesert soils are aeolian (wind-deposited) and consist of sand and siltsized particles with little or no organic content. Such soils areconsidered poor for agricultural development because they are highlyporous, which promotes rapid infiltration and seepage of irrigationwater away from the surface where it is most needed for crops. Lack oforganic material generally corresponds to low nutrient content. Manydesert areas are active aeolian environments in which wind is thedominant agent of sediment transport.

Agricultural development is severely impacted when topsoil is eroded andtransported by wind. Considering the characteristics of sandy desertsoils versus the requirements of soils for agriculture, the presentinvention includes the use of the LAB-1 derived biofilm for thetreatment of agricultural soils to improve the following soilagricultural properties: (1) improved water retention characteristics;(2) enhanced ability to establish and support plant growth; and (3)improved erosion resistance. These improvements may be obtained byadding complete or dried and pulverized biofilm, or by the applicationof LAB-1 strain in bacterial/nutrient solutions using conventional soilwatering equipment (e.g., a truck-mounted tank with sprinkler hoses orconventional irrigation systems).

The biofilm of the present invention has been used for the treatment ofsoil. The biofilm altered the soil's properties in many ways thatenhanced the soil's ability to support agriculture. These include thefollowing: (1) an improved ability of sand to retain moisture; (2) anincreased biomass in the form of polysaccharides that function as anutrient supply for plant growth; (3) improved soil cohesion; and (4)increased resistance of soil to erosion.

While this invention has been described in reference to illustrativeembodiments, this description is not intended to be construed in alimiting sense. Various modifications and combinations of theillustrative embodiments, as well as other embodiments of the invention,will be apparent to persons skilled in the art upon reference to thedescription. It is therefore intended that the appended claims encompassany such modifications or embodiments. All references cited herein arehereby expressly incorporated by reference.

What is claimed is:
 1. A process for plugging a permeable stratum,comprising the steps of a) providing into a permeable stratum, a biofilmwhich comprises an elastic exopolysaccharide which exopolysaccharide isthe exopolysaccharide produced by a bacterium which has thecharacteristics that it is Gram negative, bacilliary, about 0.2×0.8 μm,facultative anaerobe, grows between 15° and 45° C. with a temperatureoptimum of 37° C., grows between pH 4-1 1 but not at pH 2, grows in AB13medium or minimal medium, is motile, lacks a capsule, and lacks spores,and b) incubating said biofilm for an amount of time sufficient toproduce a plugged stratum.
 2. The process of claim 1, wherein theplugged stratum has a saturated hydraulic conductivity equal to orless/than 1.0×10⁻⁷ cm/sec.
 3. The process of claim 1, wherein theplugged stratum has a saturated hydraulic conductivity equal to or lessthan 1.5×10⁻⁸ cm/sec.
 4. The process of claim 1, wherein said bacteriumfurther comprises the characteristics of an antibiotic sensitivityprofile as in Table 2, a biochemistry profile as in Table 3, and acarbon utilization profile as in Table
 4. 5. The process of claim 4,wherein the bacterium is that deposited as ATCC No. PT 2500.